Profiling natural serum antibodies of non‐human primates with a carbohydrate antigen microarray

Abstract

Background: Engineering of α-Galactosyltransferase gene-knockout pigs circumvented hyperacute rejection of pig organs after xenotransplantation in non-human primates. Overcoming this hurdle revealed the importance of non-α-Gal carbohydrate antigens in the immunobiology of acute humoral xenograft rejection. Methods: This study analyzed serum from seven naïve cynomolgus monkeys (blood type O/B/AB = 3/2/2) for the intensity of natural IgM and IgG signals using carbohydrate antigen microarray, which included historically reported α-Gal and non-α-Gal carbohydrate antigens with various modifications.Results: The median (range) of IgM and IgG signals were 12.71 (7.23-16.38) and 9.05 (7.23-15.90), respectively. The highest IgM and IgG signals with narrowest distribution were from mono-and disaccharides, followed by modified structures. Natural anti-α-Gal antibody signals were medium to high in IgM (11.2-15.9) and medium in IgG (8.5-11.6) spectra, and was highest with Lac core structure (Galα1-3Galβ1-4Glc, iGb3) and lowest with LacNAc core structure (Galα1-3Galβ1-4GlcNAc). Similar signal intensities (up to 15.8 in IgM and up to 11.8 in IgG) were observed for historically detected natural non-α-Gal antigens, which included Tn antigen, T antigen, GM2 glycolipid, and Sd a antigen. The hierarchical clustering analysis revealed the presence of clusters of anti-A antibodies and was capable of distinguishing between the blood group B and AB non-human primates. Conclusions:The results presented here provide the most comprehensive evaluation of natural antibodies present in cynomolgus monkeys. K E Y W O R D S carbohydrate antigen microarray, natural serum antibodies, non-human primates, xenotransplantation GM3(Gc)−Sp − 05 CT/Sda−Sp − 05

Carbohydrate antigen microarray analysis of serum IgG and IgM antibodies before and after adult porcine islet xenotransplantation in cynomolgus macaques

“…The details of signal intensities are shown in S1 Table . The distribution of anti-carbohydrate antibody signal intensity in the sera was mostly similar to that of naïve sera in the previous report [ 18 ], but blood group A antigens (BG-A5-16, and BG-A-19) were listed in the posttransplant IgM and IgG repertoires with a wide distribution ( Fig 1 ).…”

“…We have previously performed a detailed analysis for natural IgM and IgG antibody levels of naïve cynomolgus macaques ( Macaca fascicularis ) of Mauritian origin [ 18 ]. Five male monkeys with a median age of 5.3 years (range: 4.4–5.8) and a median weight of 5.0 kg (4.4–7.6) with blood type O ( n = 2), B ( n = 2), and AB ( n = 1) were recipients of adult porcine islet xenografts.…”

“…Five male monkeys with a median age of 5.3 years (range: 4.4–5.8) and a median weight of 5.0 kg (4.4–7.6) with blood type O ( n = 2), B ( n = 2), and AB ( n = 1) were recipients of adult porcine islet xenografts. The housing conditions of the monkeys were described previously [ 18 ]. Briefly, They were housed in same-sex pairs or small groups.…”

“…Recipient monkey monitoring included daily clinical assessments by study staff, regular evaluations by veterinary staff, and weekly hematology and chemistry laboratory studies. Blood samples were taken from a totally implantable venous access port as previously described [ 18 ]. Posttransplant serum samples were collected at the time of clinical rejection (posttransplant day 142, 46, 42, 28, and 35 for monkey 13GP08, 12JP01, 13CP03, 13CP10, and 13GP10, respectively).…”

“…Identifying additional novel xenoantigens will play an important role in developing strategies for establishing long-term graft function. We have recently performed a detailed analysis of natural IgM and IgG antibody specificity and concentration from naïve cynomolgus macaques utilizing a novel carbohydrate microarray (glycan microarray), in which more than 400 glycoconjugates and carbohydrates are attached to solid supports [ 18 ]. Use of high-resolution carbohydrate microarray has the potential to elucidate the specific carbohydrate target of rejection by directly comparing naïve and posttransplant sera from individual monkeys.…”